Base editing the synapse: Modeling a complex neurological disorder in non-human primates
Authors: Benjamin L. Prosser, Ingo Helbig
Journal: Molecular Therapy (2022)

Since its discovery in 2016,1Komor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424https://doi.org/10.1038/nature17946Crossref PubMed Scopus (2585) Google Scholar base editing—the efficient chemical modification of single nucleotides—has enjoyed a blistering pace of advance alongside the broader field of gene editing. In this issue of Molecular Therapy, Lu and colleagues2Lu, Z., He, S., Jiang, J., Zhuang, L., Wang, Y., Yang, G., Jiang, X., Nie, Y., Fu, J., Zhang, X. and Lu, Y.. Base-edited cynomolgus monkeys mimic core symptoms of STXBP1 encephalopathy. https://doi.org/10.1016/j.ymthe.2022.03.001.Google Scholar take an important step forward in making these technologies applicable to human disease, reporting on the successful use of a base editor to model a complex neurological disorder in non-human primates. STXBP1-related neurodevelopmental disorder (STXBP1-NDD) represents one of the most common genetic neurodevelopmental disorders and is characterized by epilepsy and cognitive, behavioral, and motor impairments3Xian J. Parthasarathy S. Ruggiero S.M. Balagura G. Fitch E. Helbig K. Gan J. Ganesan S. Kaufman M.C. Ellis C.A. et al.Assessing the landscape of STXBP1 -related disorders in 534 individuals.Brain. 2021; 2021: awab327https://doi.org/10.1093/brain/awab327Crossref Scopus (16) Google Scholar,4Stamberger H. Nikanorova M. Willemsen M.H. Accorsi P. Angriman M. Baier H. Benkel-Herrenbrueck I. Benoit V. Budetta M. Caliebe A. et al.STXBP1 encephalopathy A neurodevelopmental disorder including epilepsy.Neurology. 2016; 86: 954-962https://doi.org/10.1212/WNL.0000000000002457Crossref PubMed Scopus (200) Google Scholar Yet, a lack of adequate model systems has hindered understanding of the underlying pathophysiology. While mouse models recapitulate certain aspects of the disease,5Kovačević J. Maroteaux G. Schut D. Loos M. Dubey M. Pitsch J. Remmelink E. Koopmans B. Crowley J. Cornelisse L.N. et al.Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy.Brain. 2018; 141: 1350https://doi.org/10.1093/brain/awy046Crossref PubMed Scopus (55) Google Scholar,6Chen W. Cai Z.-L. Chao E.S. Chen H. Longley C.M. Hao S. Chao H.-T. Kim J.H. Messier J.E. Zoghbi H.Y. et al.Stxbp1/Munc18-1 haploinsufficiency impairs inhibition and mediates key neurological features of STXBP1 encephalopathy.Elife. 2020; 9: e48705https://doi.org/10.7554/elife.48705Crossref PubMed Scopus (0) Google Scholar these models also have important limitations. The primate model generated here exhibits electrical abnormalities difficult to capture in rodents, thus offering a two-pronged advance—one for the field of base editing, and one for the translational toolkit of developmental epilepsies. Base editors evolved from the CRISPR-Cas9 system and utilize similar components for targeting specific regions of DNA. Yet, base editors apply a unique deaminase domain for DNA modification and a modified Cas9 “nickase” that cuts only one DNA strand. This allows precise, single-nucleotide edits while minimizing the unintended consequences of double-stranded breaks. The reduction in unintended, on-target editing (insertions/deletions at the site of double-strand breaks) and high efficiency of base editing even in non-dividing cells has led to considerable enthusiasm for its therapeutic potential.7Musunuru K. Moving toward genome-editing therapies for cardiovascular diseases.J. Clin. Invest. 2022; 132: e148555https://doi.org/10.1172/jci148555Crossref PubMed Google Scholar After the initial development of a cytosine base editor (CBE) for human cells in 2016, an adenine base editor (ABE) rapidly followed along with novel engineered deaminase and Cas variants that reduce off-target RNA editing and improve editing efficiency (see timeline in Figure 1). This rapid technological development manifested in the milestone application of ABEs for therapeutic gene manipulation of non-human primates in 2021, where editing of the cholesterol-regulating gene PCSK9 durably lowered cholesterol levels in cynomolgus monkeys.8Musunuru K. Chadwick A.C. Mizoguchi T. Garcia S.P. DeNizio J.E. Reiss C.W. Wang K. Iyer S. Dutta C. Clendaniel V. et al.In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.Nature. 2021; 593: 429-434https://doi.org/10.1038/s41586-021-03534-yCrossref PubMed Scopus (228) Google Scholar,9Rothgangl T. Dennis M.K. Lin P.J.C. Oka R. Witzigmann D. Villiger L. Qi W. Hruzova M. Kissling L. Lenggenhager D. et al.In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels.Nat. Biotechnol. 2021; 39: 949-957https://doi.org/10.1038/s41587-021-00933-4Crossref PubMed Scopus (112) Google Scholar The current study by Liu and collaborators2Lu, Z., He, S., Jiang, J., Zhuang, L., Wang, Y., Yang, G., Jiang, X., Nie, Y., Fu, J., Zhang, X. and Lu, Y.. Base-edited cynomolgus monkeys mimic core symptoms of STXBP1 encephalopathy. https://doi.org/10.1016/j.ymthe.2022.03.001.Google Scholar provides proof of concept for another important utility of base-editing—the generation of disease models that may closely mimic the human condition, particularly genetic neurodevelopmental disorders. STXBP1-NDD arises from heterozygous, de novo variants in the STXBP1 gene, which encodes for the pre-synaptic protein syntaxin-binding protein 1, an essential component for neurotransmitter release. Disease-causing variants are spread across the STXBP1 gene and most commonly consist of single-point, loss-of-function variants,3Xian J. Parthasarathy S. Ruggiero S.M. Balagura G. Fitch E. Helbig K. Gan J. Ganesan S. Kaufman M.C. Ellis C.A. et al.Assessing the landscape of STXBP1 -related disorders in 534 individuals.Brain. 2021; 2021: awab327https://doi.org/10.1093/brain/awab327Crossref Scopus (16) Google Scholar,5Kovačević J. Maroteaux G. Schut D. Loos M. Dubey M. Pitsch J. Remmelink E. Koopmans B. Crowley J. Cornelisse L.N. et al.Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy.Brain. 2018; 141: 1350https://doi.org/10.1093/brain/awy046Crossref PubMed Scopus (55) Google Scholar although there exists some evidence for missense variants driving dominant-negative mechanisms.10Guiberson N.G.L. Pineda A. Abramov D. Kharel P. Carnazza K.E. Wragg R.T. Dittman J.S. Burré J. Mechanism-based rescue of Munc18-1 dysfunction in varied encephalopathies by chemical chaperones.Nat. Commun. 2018; 9: 3986https://doi.org/10.1038/s41467-018-06507-4Crossref PubMed Scopus (36) Google Scholar There are at least five genetic hotspots that exhibit recurrent missense variants, with one of the most prominent located at p.Arg292. Variants at this locus are associated with electroclinical syndromes typical of the disorder, with prominent neonatal seizures and neurodevelopmental differences.3Xian J. Parthasarathy S. Ruggiero S.M. Balagura G. Fitch E. Helbig K. Gan J. Ganesan S. Kaufman M.C. Ellis C.A. et al.Assessing the landscape of STXBP1 -related disorders in 534 individuals.Brain. 2021; 2021: awab327https://doi.org/10.1093/brain/awab327Crossref Scopus (16) Google Scholar Lu and colleagues capitalize on the suitability of this genomic location for base editing to introduce a C to T substitution that creates the R292H mutation following intracytoplasmic sperm injection of a CBE in cynomolgus monkey embryos. This is relevant as the technology is currently limited to transition edits of C and A nucleotides. After implantation and pregnancy, two live monkeys were born, while five fetuses were aborted. Live monkeys exhibited heterozygous R292H mutations in the majority of cells, thus reflecting the patient genotype, while aborted fetuses exhibited close to homoyzygous mutation rates, consistent with the homozygous mutation resulting in fetal lethality. This low yield of viable monkeys permits more of a case report than systematic study of disease phenotypes—a caveat that must be applied to each discussion point below, and also underscores the challenging ethical considerations that surround such studies. The cynomolgus monkey (macaque) has enabled numerous milestones in neurobiological research—particularly for cognitive neuroscience—which the authors document admirably. The current work also emphasizes potential advantages of the macaque for examining complex circuit abnormalities and electroencephalogram (EEG) patterns seen in patients but that are difficult to recapitulate in rodents—which is perhaps unsurprising given the cross-species differences in neuronal populations and pathways. In this light, the EEG analysis of the STXBP1 p.R292H monkey offers interesting points for discussion. Mice heterozygous for STXBP1 are useful, well-documented models that recapitulate several important human phenotypes including impaired cognitive performance and epileptic activity, which specifically manifest as spike-wave discharges5Kovačević J. Maroteaux G. Schut D. Loos M. Dubey M. Pitsch J. Remmelink E. Koopmans B. Crowley J. Cornelisse L.N. et al.Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy.Brain. 2018; 141: 1350https://doi.org/10.1093/brain/awy046Crossref PubMed Scopus (55) Google Scholar,6Chen W. Cai Z.-L. Chao E.S. Chen H. Longley C.M. Hao S. Chao H.-T. Kim J.H. Messier J.E. Zoghbi H.Y. et al.Stxbp1/Munc18-1 haploinsufficiency impairs inhibition and mediates key neurological features of STXBP1 encephalopathy.Elife. 2020; 9: e48705https://doi.org/10.7554/elife.48705Crossref PubMed Scopus (0) Google Scholar However, these features do not translate directly to clinical features seen in patients with STXBP1-NDD. For example, certain severe electrophysiological abnormalities such as suppression-burst patterns that are seen in a subset of individuals are prominent in STXBP1 p.R292H macaques yet are not observed in rodents. Further, the commonly prescribed anti-epileptic medication levetiracetam successfully eliminates the prominent spike-wave discharges in STXBP1 heterozygous mice5Kovačević J. Maroteaux G. Schut D. Loos M. Dubey M. Pitsch J. Remmelink E. Koopmans B. Crowley J. Cornelisse L.N. et al.Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy.Brain. 2018; 141: 1350https://doi.org/10.1093/brain/awy046Crossref PubMed Scopus (55) Google Scholar yet is generally ineffective at maintaining seizure freedom in patients with STXBP1-NDD,3Xian J. Parthasarathy S. Ruggiero S.M. Balagura G. Fitch E. Helbig K. Gan J. Ganesan S. Kaufman M.C. Ellis C.A. et al.Assessing the landscape of STXBP1 -related disorders in 534 individuals.Brain. 2021; 2021: awab327https://doi.org/10.1093/brain/awab327Crossref Scopus (16) Google Scholar hinting at important differences where information from model systems may be potentially misleading. Intriguingly, treatment of the STXBP1 p.R292H monkey with levetiracetam reduced seizure-like spikes as in rodents yet had no effect on suppression-burst EEG patterns. This highlights potential differences in non-specific treatment responses seen in rodent models compared with more accurate reflections of the disease mechanisms in non-human primates. These observations suggest that macaques may better model the developmental encephalopathy component that is difficult to emulate in rodents, a finding worth closer examination in STXBP1 and additional models of developmental and epileptic encephalopathies. Macaque models of neurological disorders also present other, more obvious advantages for therapeutic testing. STXBP1-NDD is a strong candidate for gene-replacement therapy. The condition is a monogenic, loss-of-function disorder where the gene of interest fits the carrying capacity of existing adeno-associated viral (AAV) vectors. Numerous such programs are underway for a variety of genetic encephalopathies, often with positive indications from AAV-mediated gene replacement in rodents. Yet, delivery of cargo via AAV to the rodent brain appears a far simpler task than efficient biodistribution to primate neurons, and the same viral vectors may not be compatible. An STXBP1 macaque model has clear advantages for testing the safety and efficacy of gene-replacement strategies targeting neuronal populations. Another major challenge of gene therapy is determining which specific neuronal populations need to be targeted for optimal therapeutic results. While the current study does not answer this question, it does offer intriguing food for thought prompted by single-cell RNA sequencing (scRNA-seq) of the prefrontal cortex of an STXBP1-NDD and wild-type (WT) monkey. After clustering identified several subtypes of cortical excitatory neurons, interneurons, astrocytes, and other glial populations, the authors identify a specific subtype of excitatory neuron that warrants deeper interrogation. These neurons exhibited the highest expression of STXBP1 and its synergistic partner STX1A and were selectively lost in the STXBP1-NDD monkey. This provocatively suggests that preferential dysfunction or loss of certain neuronal subpopulations may contribute to disease; however, given the limited throughput of the macaque system, these results require further validation. In sum, the work of Lu et al. represents both a marked advance and a clear reminder of how far we must go: base editing is a powerful tool, but it still presents considerable challenges. For example, the 100-fold higher incidence of off-target single-nucleotide variants in edited monkeys relative to WT reinforces a common concern of genome editing. It can be expected that many of these limitations will be overcome even as they are revealed—such is the pace in genome editing. Regarding the use of base editing to generate primate models of neurodevelopmental disorders, the study by Lu et al. tantalizes with just how much we might learn and, conversely, could be missing with a highly skewed emphasis on rodent work. Yet, challenges of feasibility, access, viability, and ethics all cloud the assessment of just where such models should fit in our pre-clinical pipelines. Given the rate of development inherent to such promising technologies, much of this cloudiness stands to be cleared in the upcoming years.